Development of selective blockers for Ca2+-activated Cl- channel using Xenopus laevis oocytes with an improved drug screening strategy

<p>Abstract</p> <p>Background</p> <p>Ca²⁺-activated Cl^-channels (CaCCs) participate in many important physiological processes. However, the lack of effective and selective blockers has hindered the study of these channels, mostly due to the lack of good assay system. Here, we have developed a reliable drug screening method for better blockers of CaCCs, using the endogeneous CaCCs in <it>Xenopus laevis </it>oocytes and two-electrode voltage-clamp (TEVC) technique.</p> <p>Results</p> <p>Oocytes were prepared with a treatment of Ca²⁺ionophore, which was followed by a treatment of thapsigargin which depletes Ca²⁺stores to eliminate any contribution of Ca²⁺release. TEVC was performed with micropipette containing chelerythrine to prevent PKC dependent run-up or run-down. Under these conditions, Ca²⁺-activated Cl^-currents induced by bath application of Ca²⁺to oocytes showed stable peak amplitude when repetitively activated, allowing us to test several concentrations of a test compound from one oocyte. Inhibitory activities of commercially available blockers and synthesized anthranilic acid derivatives were tested using this method. As a result, newly synthesized <it>N</it>-(4-trifluoromethylphenyl)anthranilic acid with trifluoromethyl group (-CF₃) at <it>para </it>position on the benzene ring showed the lowest IC₅₀.</p> <p>Conclusion</p> <p>Our results provide an optimal drug screening strategy suitable for high throughput screening, and propose <it>N</it>-(4-trifluoromethylphenyl)anthranilic acid as an improved CaCC blocker.</p

C-terminally mutated tubby protein accumulates in aggresomes

The tubby protein (Tub), a putative transcription factor, plays important roles in the maintenance and function of neuronal cells. A splicing defect-causing mutation in the 3&apos;-end of the tubby gene, which is predicted to disrupt the carboxy-terminal region of the Tub protein, causes maturity-onset obesity, blindness, and deafness in mice. Although this pathological Tub mutation leads to a loss of function, the precise mechanism has not yet been investigated. Here, we found that the mutant Tub proteins were mostly localized to puncta found in the perinuclear region and that the C-terminus was important for its solubility. Immunocytochemical analysis revealed that puncta of mutant Tub co-localized with the aggresome. Moreover, whereas wild-type Tub was translocated to the nucleus by extracellular signaling, the mutant forms failed to undergo such translocation. Taken together, our results suggest that the malfunctions of the Tub mutant are caused by its misfolding and subsequent localization to aggresomes.ope

Accuracy of analytical approaches to thermal response test interpretation

Thermal conductivity obtained via thermal response test (TRT) is one of the essential parameters for the design of shallow geothermal systems, especially a borehole heat exchanger (BHE). During TRT analysis, several factors (e.g., start time of analysis and test duration) could influence the estimated thermal conductivity. In addition, the influence of the factors may be different depending on the characteristics of the borehole and ground. This study investigated the effects of the start time and test duration through numerically generated TRT data under a diversity of environment and BHE information such as the thermal conductivity of grouting and ground. The generated numerical data were analyzed with three analytical approaches having different assumptions about the heat source to interpret the sensitivity of factors in the TRT analysis. By conducting the analytical sensitivity analysis, the importance of determining the appropriate start time and test duration could be emphasized when designing the test

Computed tomography–assessed presarcopenia and clinical outcomes after laparoscopic surgery for rectal cancer

Purpose Previous studies have reported that presarcopenia negatively affects rectal cancer treatment. However, most studies have analyzed patients including majority of open surgery, and the association between presarcopenia and clinical outcomes after laparoscopic rectal cancer surgery remains unclear. This study aimed to evaluate the impact of presarcopenia on the clinical and oncological outcomes after laparoscopic rectal cancer surgery. Methods Three hundred and one patients undergoing laparoscopic rectal cancer surgery between December 2009 and May 2016 were enrolled. Body composition was assessed using computed tomography by measuring the muscle and fat areas at the third lumbar (L3) vertebra. The L3 skeletal muscle area was used to calculate the skeletal muscle index and evaluate presarcopenia. Results Presarcopenia was more common in older (≥70 years, P=0.008) or female patients (P=0.045). Patients with presarcopenia had decreased skeletal muscle area (P<0.001), lower hemoglobin level (P=0.034), longer time to first flatus (P<0.001), and more frequent surgical site infection (P=0.001). However, survival rates were not significantly different between those with and without presarcopenia. Conclusion Computed tomography-assessed presarcopenia was associated with delayed functional recovery and increased surgical site infection, although it was not revealed as a prognostic factor for oncological outcomes

Effect Of Nitric-Oxide Post-Oxidation Annealing On High-Temperature Oxidized 4H SiC.

Metal-Oxide-Semiconductor FETs using 4H-SiC have been investigated intensively because 4H-SiC semiconductor has excellent physical properties for power-device applications

Application of CRISPR-Based C-to-G Base editing in rice protoplasts

Recently, new types of base editors, C-to-G base editors (CGBEs), that enable cytosine transversions that are unachievable with cytosine base editors (CBEs) and adenosine base editors (ABEs), have been developed in human cells. However, despite their importance in crop genome editing, the efficacy of CGBEs has not yet been extensively evaluated. In our study, based on the previously reported plant-compatible CBE and human CGBE, we demonstrated that our monocot plant-compatible CGBEs (PcCGBEs) enable cytosine transversions (C-to-G) in rice protoplasts. For all targets tested, PcCGBEs (monocot plant-compatible CGBEs) appeared to have substantial levels of C-to-G editing activity. PcCGBE showed a much higher C-to-G base editing activity and C-to-G specificity among C-to-D conversions than the mini-version of PcCGBE. Our demonstration of PcCGBE could provide a platform for the further development of enhanced CGBEs for reliable application as a new crop breeding technology.This work was supported by the Creative-Pioneering Researchers Program of Seoul National University, National Research Foundation of Korea (NRF) Grant (2019R1F1A1046305), and New Breeding Technologies Development Program (PJ016542) through the Rural Development Administration (RDA), Republic of Korea

Characteristics of Sawdust and Cocopeat Beddings, and Their Usefulness According to the Fan and Pen Location for Rearing Hanwoo Cattle

This study was designed to examine the characteristics of sawdust and cocopeat bedding materials, including physicochemical properties (Exp. I) and on-farm trial (Exp. II). In Exp. I, the proportion of particle size was in the order of sawdust> cocopeat India>cocopeat Vietnam (pcocopeat Vietnam>sawdust (p<0.05), thus cocopeat treatments showed 4.4 times higher bedding cost than sawdust. The water absorption rates were 702.0% in cocopeat India, 678.3% in cocopeat Vietnam, and 444.0% in sawdust, showing cocopeat had approximately 1.5 times higher water absorption rate than sawdust. Moisture evaporation rates after 12 h of air blowing (2.00 m/s) were higher (p<0.05) in cocopeat Vietnam (80.4%) than sawdust (71.2%) and cocopeat India (72.8%). In vitro ammonia emissions were higher (p<0.05) in sawdust (2.71 mg/m2/h) than cocopeat India (1.59 mg/m2/h) and Vietnam (1.22 mg/m2/h), and total ammonia emissions were higher (p<0.05) in sawdust (37.02 mg/m2) than cocopeat India (22.51 mg/m2) and Vietnam (13.60 mg/m2). In Exp. II, an on-farm trial was conducted with 48 Hanwoo cattle in 16 pens using the same bedding materials as in Exp. I, with fan (blowing 2.00 m/s) and no fan treatments, and feed bunk side (FB) and water supply side (WS) within a pen (4.5 m, width×9.0 m, length). Beddings were replaced with fresh bedding materials when moisture concentrations were over 65%. No interactions among treatments were detected for moisture concentration and increment rates, and ammonia concentrations, but a significant effect was observed (p<0.01) for each of the treatments. Both concentrations and increment rate of moisture were higher (p<0.01) in the beddings without fan than with fan. Moisture concentrations and increment rate within a pen were also higher (p<0.01) in FB than WS. Thus, the whole no-fan-FB and sawdust-fan-FB were replaced with fresh bedding material between 4 to 5 experimental weeks. The ammonia concentrations and pH of beddings were not significantly different among treatments. Therefore, using cocopeat bedding with a blowing fan can extend twice the bedding utilization period, and WS within a pen showed twice the bedding-life compared to FB. Despite the outstanding characteristics of cocopeat compared with sawdust, using cocopeat as an alternative for sawdust bedding is not recommended for cattle management, considering it has 4.4 times higher bedding cost and a dust production problem